The use of recombinant Baculoviruses as expression vectors is well known. Typically, the use of recombinant Baculovirus vectors involves the construction and isolation of recombinant Baculoviruses in which the coding sequence for a chosen gene is inserted behind the promoter for a nonessential viral gene, polyhedrin. A number of Baculovirus expression vectors based on the polyhedrin promoter have been previously described. Smith et al., Mol. Cell. Biol. 3:2156-2165 (1985); Posse, Virus Res. 5:43-59 (1986); and Matsuura, J. Gen. Virol. 68:1233-1250 (1987).
One advantage of the Baculovirus vectors over bacterial and yeast expression vectors includes the expression of recombinant proteins that are essentially authentic and are antigenitally and/or biologically active. In addition, Baculoviruses are not pathogenic to vertebrates or plants and do not employ transformed cells or transforming elements as do the mammalian expression systems. Although mammalian expression systems result in the production of fully modified, functional protein, yields are often low. E. coli systems result in high yields of recombinant protein but the protein is not modified and may be difficult to purify in a nondenatured state.
The usual host cell for a Baculovirus vector is Sf9, a clonal isolate established from Spodoptera frugiperda, commonly known as the fall army worm. Usually, the use of a Baculovirus vector in combination with Sf9 results in the production of proteins that are essentially authentic and are antigenitally and/or biologically active. Expression in Baculovirus-infected insect cells has the advantage of generating large amounts of protein closely related to its native counterpart and is often much easier to purify. However, further investigations have suggested that the Sf9 cells and mammalian cells exhibit some differences in the basic mechanism of protein glycosylation. In particular, one major difference between recombinant proteins produced in insect cells and those derived from mammalian cells is the extent and type of oligosaccharide processing.
Sf9 cells used for Baculovirus expression are capable of adding N-glycosylated carbohydrate chains to proteins and in some cases process them to an endo-H-resistant form.
The addition of carbohydrate to a protein is important because carbohydrate often plays a major role in the overall structure and function of a protein. More specifically, carbohydrates are responsible for blood group antigenicity, controlling events in growth and differentiation, and they also contribute to the protection of proteins from degradation. In addition, oligosaccharides may maintain protein conformation in a biologically active state.
There are several examples in the literature of proteins that are glycosylated or secreted efficiently early in virus infection (24 hours p.i.), but fail to be glycosylated or secreted late in infection (after 48 hours p.i.) when the polyhedrin promoter of the Baculovirus vector is most active. Murphy et al., Gen. Anal. Tech. Appl. 7:160-171 (1990); Jarvis et al., Mol. Cell. Biol. 9:214-223 (1989); and Bailey et al., Virology 169:323-331 (1989). In these uses, poor yields of recombinant product were obtained compared with the amount of polyhedrin produced in wild type Baculovirus infection. For example, Jarvis et al. examined the pathway of protein glycosylation and secretion in Sf9 cells using human tissue plasminogen activator (TPA) as a model. Although Jarvis et al. report that the TPA expressed by Sf9 was both N-glycosylated and secreted, the relative efficiency of secretion decreased dramatically with time of infection. Jarvis et al.'s results led them to conclude that the Sf9 host cell secretory pathway is therefore somehow compromised during the later stages of Baculovirus infection.
A need therefore exists for a Baculovirus vector that will allow for the production of proteins that are glycosylated and secreted in the late term of infection when its promoter is most active.
Some success in increasing yields of proteins directed to the endoplasmic reticulum has been obtained by manipulating the signal peptide of the recombinant protein. When the honeybee mellitin signal peptide was fused to the plant papain precursor it was secreted over five times more than the wild type protein containing the plant signal peptide (Tessier et al., Gene 98:177-183 (1991)). However, the use of a consensus signal peptide based on a survey of eukaryotic signal peptides by von Heijne (von Heijne, G., Nucl. Acids Res. 11:4683-4691 (1986)) did not result in significantly more secretion of chimeric plasminogen activator proteins (Devlin, J. J., et al., Biotechnology 7:286-292 (1989)). This application describes the construction of two secretion vectors containing the egt and p67 signal peptides and the use of these vectors to express and secrete the HIV-1 gp120 protein from insect cells.